Alexander disease is a rare and typically fatal neurodegenerative disease that results from heterozygous mutations in the gene encoding the type III intermediate filament protein GFAP. The pathological signature of the disorder is the Rosenthal fiber, a cytoplasmic inclusion containing intermediate filaments and small heat shock proteins that accumulates in astrocytes throughout the CMS. Prior investigations by our groups have let to the acceptance of GFAP mutations as the cause for nearly all cases of Alexander disease, and the rapid translation of this information to clinical practice as the standard for diagnosis. However, the mechanisms by which GFAP mutations cause astrocyte dysfunction and disease remain unclear. Based on results obtained during the previous grant period we have developed a hypothesis to guide experiments that emphasizes expression of elevated levels of GFAP in concert with induction of a cellular stress response and formation of inclusions as key elements of pathogenesis. The goals of this Program Project are to investigate molecular mechanisms and functional consequences of the stress response, to explore biochemical composition and downstream effects of inclusion body formation, and to identify and characterize genetic modifiers of disease phenotype. Our studies span genetic, biochemical, cellular, physiological, and morphological approaches to these questions. The Program will link five laboratories;three of these continue from the previous grant period, and two new groups join that bring novel approaches and techniques (physiology and Drosophila). The Program will promote a focussed effort on the role of glial filament dysfunction in disease, by fostering sharing of reagents, animals, and results among the five labs, through cross-fertilization of ideas, and by regular communication and meetings among laboratory members. These studies promise novel insights into the role of glial filaments in the cell biology of astrocytes, and the role of astrocytes in brain function and disease. PROJECT 1 Principal Investigator: Michael Brenner Title: Biochemistry and Genetics of Alexander Disease Description (provided by applicant): Glial fibrillary acidic protein (GFAP) is a structural protein found almost exclusively in astrocytes. Our laboratory recently found that mutations in the coding region of the GFAP gene cause Alexander disease (AxD), a rare but usually fatal disorder of the central nervous system. This disease is characterized by the presence of protein aggregates which have GFAP as a primary constituent. The purpose of this proposal is to develop additional information about the mechanism by which the GFAP mutations cause AxD. In Aim 1 we will determine the composition of the RFs to obtain clues for the disease mechanism, an approach that has proved fruitful for other protein aggregate disorders. The aggregates will be partially purified, and their protein components identified by mass spectrometry. In Aim 2 we will determine if the GFAP is abnormally deiminated or phosphorylated, two modifications of GFAP known to affect its polymerization and to be present in other neurodegenerative disorders. In Aim 3 we will also determine whether the mutant form specifically accumulates in the aggregates. This will test the hypothesis that mutant GFAP is not toxic per se, but produces disease by causing GFAP accumulation.